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Bulk RNA-seq, small RNA-seq, and miRNA pulldown profiling of H19 RNA editing and miR-675-3p retargeting in mouse hematopoietic cells

ABSTRACT: Adenosine-to-inosine RNA editing can alter RNA structure, processing, and target recognition, but its physiological functions during hematopoietic stem cell development remain incompletely understood. This study investigates how developmental editing of the imprinted long noncoding RNA H19 regulates hematopoietic stem cell quiescence through the biogenesis and target specificity of miR-675-3p. The deposited datasets comprise conventional bulk RNA sequencing, small RNA sequencing, and biotinylated miRNA pulldown RNA sequencing from mouse hematopoietic stem and progenitor cells and related cell-line models. Bulk RNA-seq was performed in 32D-LV-H19 cells expressing empty vector, wild-type ADAR1 p110, or catalytically inactive ADAR1 p110 E861A; primary mouse c-Kit-positive cells expressing empty vector, wild-type editable H19-A, the H19-G edited mimic, or the editing-resistant H19-T allele; poly(I)-treated Ki67-eGFP-negative LT-HSCs carrying H19-A, H19-G, or H19-T alleles; and steady-state Ki67-eGFP-negative and Ki67-eGFP-positive LT-HSCs. Small RNA-seq was used to quantify miRNA production in 32D-LV-H19 and primary c-Kit-positive cells under the corresponding ADAR1- and H19-dependent experimental conditions. In parallel, biotinylated miRNA pulldown RNA-seq was performed in 32D cells transfected with scrambled-control, unedited wild-type miR-675-3p, or edited miR-675-3p mimics. Input and pulldown fractions were sequenced to identify transcripts preferentially associated with each miRNA species. Together, these datasets enable integrated analysis of H19 editing-dependent transcriptional changes, miR-675-3p biogenesis, and editing-induced miRNA target retargeting in hematopoietic cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE341465 | GEO | 2026/08/04

REPOSITORIES: GEO

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Project description:Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Examination of the effect of ADAR2 on mature miRNA abundance and sequence in adult mouse brain.

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Bulk RNA-seq, small RNA-seq, and miRNA pulldown profiling of H19 RNA editing and miR-675-3p retargeting in mouse hematopoietic cells

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